The rising incidence of disease caused by Mycobacterium tuberculosis, and the emergence of multidrug-resistant M. tuberculosis strains has become an international concern. These issues have led the World Health Organization to declare tuberculosis a global health emergency, a distinction never accorded another disease. Diagnostic assays currently available are severely limited in their ability to differentiate latent M. tuberculosis infection from vaccination with M. bovis BCG. Identification of acid-fast bacilli by sputum microscopy or examination of a chest-X-ray for characteristic changes are the only methods for rapid diagnosis of tuberculosis in clinical use. However, these methods are time consuming, insensitive, and of little use in children. Recent advances in mycobacterial genetics have permitted the development of many promising new strategies for diagnosis and for drug-susceptibility testing of M. tuberculosis. Several of these technologies, including luciferase reporter phages, PCR-SSCP, and Qbeta replicase-amplified probes, should be applicable to direct use in clinical assays. The experiments outlined in this proposal will develop these novel methods for rapid diagnosis and drug-susceptibility testing and will evaluate their usefulness in clinical settings.